Genes that improve tolerance to lignocellulosic toxins when overexpressed in yeast

ABSTRACT

The present invention provides isolated gene sequences useful in increasing lignocellulosic toxin tolerance in yeast. Such engineered yeast are useful in methods of biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast with increased lignocellulosic toxin tolerance are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.62/382,535, filed Sep. 1, 2016, and hereby incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under DE-FCO2-07ER64494awarded by the US Department of Energy. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to the production of biofuel. Moreparticularly, the present invention relates to genes that improvetolerance to lignocellulosic toxins when overexpressed in Saccharomycescerevisiae.

BACKGROUND OF THE INVENTION

Biomass is made up of complex chemicals, and the processes of freeingsugars from the lignocellulosic complexes creates a variety of moleculesthat prove to be toxic to micro-organisms used for fermentation. Beyondacute toxicity, the fermentation conditions themselves can provestressful to the organisms negatively affecting ethanol yield. Some ofthese hydrolysate toxins include amides, weak acids, and aldehydes thathave synergistic interactions with other stresses in hydrolysate,including acetate and high osmolarity.

Lignocellulosic plant material is a sustainable and renewable source ofbiomass for bioenergy and biochemical production. Plant cellulose andhemicellulose harbor significant concentrations of sugars that can beused to produce desired compounds through microbial fermentation. Inrecent years, several technologies have been developed to hydrolyzeplant biomass in order to release monomeric sugars (1, 2). For mosttypes of chemical pretreatment, the resulting hydrolysate contains highsugar concentrations, and thus high osmolarity, and also toxic compoundssuch as weak acids, furans, and phenolics that are generated as abyproduct of chemical hydrolysis. These hydrolysate toxins (HTs) areknown to inhibit microbial growth and fermentation; however, themechanisms of stress tolerance remain unclear for many of thesecompounds (3-5). Because removal of these inhibitors from thehydrolysate is expensive (6), a focus is to utilize inhibitor-tolerantmicroorganisms to produce biofuels and chemicals from plant biomass inan economically viable way.

One approach to this problem is to generate hydrolysate-tolerantmicrobes by engineering stress tolerance based on the mechanism of toxinaction. Most studies elucidating inhibitory mechanisms have focused onindividual toxins applied in isolation and have established the effectsof such toxins. For example, weak acids such as acetic, formic, andlevulinic acids inhibit cell growth and fermentation by mechanisms knownas weak acid uncoupling and intracellular anion accumulation (7). Weakacids protonated at low pH can diffuse across the plasma membranewhereupon they dissociate to decrease cytosolic pH (8) and consequentlystimulate plasma membrane ATPases that consume ATP to pump protons outof the cell (9, 10). Furans such as 5-hydroxymethyl furfural (HMF) andfurfural are also common inhibitors found in hydrolysate, formed by thedegradation of xylose and glucose, respectively (7). Furan derivativesare thought to decrease ethanol production by directly inhibitingalcohol dehydrogenase (ADH), pyruvate dehydrogenase (PDH) and aldehydedehydrogenase (ALDH) enzymes (11). In addition, furfural causes theaccumulation of reactive oxygen species that broadly damage membranes,DNA, proteins, and cellular structures (12). Cells respond by reducingfurans to less inhibitory compounds at the expense of NAD(P)+ reduction;thus the combined presence of furfural and HMF limits cell division andbiofuel production (13, 14). Among other inhibitors, phenolics are themost diverse and the least well understood. These compounds are formedduring lignin breakdown, and thus their concentrations and identitiesmainly depend on the source of plant biomass (4, 15). Phenolic compoundsexert considerable inhibitory effects by causing the loss of membraneintegrity (16, 17), decreasing cellular ATP (18, 19), causing oxidativedamage (17), inhibiting de novo nucleotide biosynthesis (20) andinhibiting translation (21). While the effects of individual toxins arebecoming clear in some cases, the compounded effects of multiple toxinsin hydrolysate are poorly understood (22, 23). Compounded stress isespecially important to consider, since microbes encounter multipleinhibitors at the same time during industrial fermentation oflignocellulosic hydrolysates.

In view of the current state of the biofuel industry, particularlyethanol production based on lignocellulosic feedstocks, it can beappreciated that identifying genes related to enhanced biofuelproduction is a substantial challenge in the field. Accordingly, a needexists in the field to identify additional genes that influence biofuelproduction in yeast, and consequently engineer recombinant strains ofyeast capable of increased biofuel yields from commonly-availablefeedstocks, including lignocellulosic plant material.

SUMMARY OF THE INVENTION

The present invention is largely related to the inventors' researchefforts to better understand tolerance to lignocellulosic toxins byyeast in the context of biofuel production. With this goal in mind, theinventors utilized high throughput competitive library screening toidentify genes involved in lignocellulosic toxin tolerance. Thisapproach implicated a variety of apparently novel genes and processes intoxin tolerance. Several of these genes significantly improvedlignocellulosic toxin tolerance when engineered in S. cerevisiae.

Based on the inventors' substantial efforts, the present inventionprovides, in a first aspect, a recombinant vector comprising: (a) thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or (b)a nucleotide sequence which hybridizes under stringent conditions to SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3 or to a fully complementarynucleotide sequence thereof; and (c) a promoter operably-linked to thenucleotide sequence of (a) or (b); wherein overexpression in yeast ofsaid nucleotide sequence provides increased tolerance to lignocellulosictoxins relative to a control yeast lacking overexpression of thenucleotide sequence.

In certain embodiments, the vector includes heterologous nucleotidesequences that stably maintain the vector at a high copy number whentransformed into yeast.

In alternate embodiments, the promoter is a heterologous promoter, asopposed to a native promoter.

In another aspect, the invention encompasses a recombinant yeastcomprising a recombinant vector as described and claimed herein.

The recombinant yeast is preferably of the genus Saccharomyces, morepreferably Saccharomyces cerevisiae.

In some embodiments, the recombinant vector is an extrachromosomalvector stably maintained in the recombinant yeast. In alternativeembodiments, the recombinant vector is integrated into a chromosome ofthe recombinant yeast.

In yet another aspect, the invention is directed to a method forproducing biofuel by fermentation of a lignocellulosic plant material inyeast, comprising: (a) culturing under biofuel-producing conditions arecombinant yeast according to the invention; and (b) isolating biofuelproduced by said recombinant yeast.

The invention further provides a method for producing biofuel byfermentation of a lignocellulosic plant material in yeast, comprising:(a) culturing under biofuel-producing conditions a recombinant yeasttransformed with a recombinant nucleic acid that overexpresses anadenylylsulfate kinase (MET14), protein folding protein foldingco-chaperone (MDJ1), or C3 sterol dehydrogenase (ERG26); and (b)isolating biofuel produced by said recombinant yeast. The recombinantyeast is preferably Saccharomyces cerevisiae.

In certain embodiments, the recombinant nucleic acid is contained in arecombinant vector that is maintained at a high copy number in therecombinant yeast.

Methods of use according to the present invention preferably utilizelignocellulosic plant material that is an ammonia fiber explosion(AFEX)-treated lignocellulosic plant material.

Yet another aspect of the invention provides a recombinant Saccharomycescerevisiae strain, comprising: (a) an isolated nucleotide sequenceencoding and overexpressing an adenylylsulfate kinase (MET14), proteinfolding protein folding co-chaperone (MDJ1), or C3 sterol dehydrogenase(ERG26); (b) or a nucleotide sequence which hybridizes under stringentconditions to said isolated nucleic acid, or to a fully complementarynucleotide sequence thereof; wherein the isolated nucleotide sequence iscontained in an extrachromosomal vector maintained at a high copy numberin the strain, and said strain exhibits increased tolerance tolignocellulosic toxins relative to a control strain lacking the isolatednucleotide sequence.

As can be appreciated, the present invention contemplates the use ofrecombinant yeast as described and claimed herein in the production ofbiofuel, including certain exemplary recombinant S. cerevisiae strainsspecifically identified in this disclosure.

This invention provides the advantage over prior biofuel-producingtechnologies in that embodiments of the invention utilize or are basedon a robust recombinant DNA approach that provides yeast strains withappreciably increased tolerance to lignocellulosic toxins. Otherobjects, features and advantages of the present invention will becomeapparent after review of the specification, claims and drawings. Thedetailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates strain-specific differences in HT tolerance. HTresistance scores were calculated as outlined in Materials and Methodsfor 79 strains. (A) HT scores measured in aerobic and anaerobicconditions are highly correlated. (B) The distribution of aerobic HTscores across all strains, where each score represents the average oftwo biological duplicates for each strain. (C) The distribution of HTscores for each of six lineages: sake, West African (WA), North American(NA), Malaysian (MA), Vineyard/European (V/E), and Mosaic (MOS). (D) Theaverage and standard deviation of HT resistance scores for each of sixstrains chosen for further analysis.

FIG. 2 depicts expression responses to SynH versus rich lab media. Shownare 2,073 differentially expressed genes identified by the linear model,as expressed in strain K11 (Sake), NCYC3290 (WA), Y7568 (MOS), YPS128(NA), and UWO.SO5.22-7 (MA). Each row represents expression of a givengene and each column represents each of two biological replicates foreach strain. Yellow indicates higher expression in the denoted straingrowing in SynH versus YPD and blue represents lower expression in SynHcompared to YPD, with fold-change according to the key. The data wereorganized by hierarchical clustering. Functional enrichments wereassessed for each cluster, and those that passed a bonferroni correctedp<0.01 included ergosterol biosynthesis (A), protein synthesis genesnormally repressed in the ESR (B), aerobic respiration (C), Gcn4 genetargets (D), sulfate assimilation (E), genes normally induced in the ESRalong with Sko1 targets (F), and ribosome biogenesis genes normallyrepressed in the ESR (G).

FIG. 3 illustrates expression differences for key groups of genes.Transcriptome differences across strains and media for specific geneclusters. Each histogram represents the average expression level (Log 2RPKM values, see Methods) of specified genes as measured in twobiological replicates for strain K11 (sake), NCYC3290 (WA), Y7568 (MOS),YPS128 (NA), and UWO.SO5.22-7 (MA). Gene clusters were selected based onhierarchical clustering of the various datasets. (A) 27 genes enrichedfor Sko1 targets, (B) 317 genes enriched for ergosterol biosynthesisgenes, (C) 27 genes enriched for sulfate assimilation genes, (D) 236genes involved in aerobic respiration, (E) 50 genes enriched for targetsof Gcn4. An asterisk indicates a significant difference in expressionfor that gene group in SynH HTs versus YPD, and a circle indicatessignificant differences in expression in SynH versus SynH −HTs (p<0.05,T-test across all genes in each group).

FIG. 4 demonstrates low pH exacerbates the effects of all HT classes.Growth rate was calculated for cells growing in SynH and SynH −HTs at pH4.5, 5.0, and 5.5 in (A) HTsensitive strain K11 (Sake) and (B)HT-resistant strain YPS128 (NA). The average and standard deviation ofgrowth rates measured in four biological replicates is shown.Statistically significant differences for each HT group at pH 4.5 versus5.0 are shown with an asterisk, and differences between pH 5.0 versus5.5 are indicated with a diamond (p<0.01, T-test)

FIG. 5 illustrates NAD levels change in response to HT exposure. (A) Theaverage and standard deviation of total NAD+/H and (B) the ratio of NAD+to NADH are shown for HT-sensitive K11 (Sake) and HT-resistant YPS128(NA). Data represent the average of biological triplicates and asterisksindicate statistical differences between SynH and SynH −HTs (*p<0.05,**p<0.01, Ttest).

FIG. 6 illustrates identifying expression differences that correlatewith HT resistance. Boxplots showing the distribution of relativetranscript abundances (measured in each strain and compared to the meanexpression of that gene across all strains). Shown are (A) 253 geneswhose transcript abundance are negatively correlated and (B) 32 geneswhose abundance is positively correlated with strain resistant scores.Strains are organized according to least (left) to highest (right)resistance.

FIG. 7 depicts how gene overexpression affects HT tolerance. (A, B) Thenumber of genes whose overexpression affected strain fitness (FDR<0.01,see Methods) is shown. (A) Genes that increased and (B) decreasedfitness in YPS128 (NA), NCYC3290 (WA) or K11 are represented in the Venndiagram. (C) Final cell density after 24 h growth of denoted strains andoverexpression constructs for cells growing in synthetic complete mediumwith high sugar content and HTs. Measurements represent the average andstandard deviation of biological triplicates. Asterisks indicatestatistical differences between Empty vector and gene overexpression(*p<0.01, Ttest).

FIG. 8 shows how iron supplementation benefits NCYC361. Shown are theaverage and standard deviation of the doubling times measured for eachstrain in YPD and in YPD supplemented with 100 mg/L of iron sulfate. Thedoubling time was faster in NCYC361 when medium was supplemented withiron (p<0.0008, T-test) but not significantly different for any otherstrain. Data represent three biological replicates.

FIG. 9 illustrates how deleting GCN4 reduces growth in both SynH −HTsand SynH. Final cell density after 24 hour growth in each medium wasmeasured for HT-resistant strain YPS128 and YPS128 gcn4D::KanMX. Shownis the average and standard deviation of two biological replicates inSynH −HTs and SynH.

FIG. 10 shows how sensitive strains showed higher induction of ESRgenes. The average transcript abundance (log 2(RPKM) values) of genesinduced in the ESR are shown for listed strains.

FIG. 11 depicts strain-specific responses to gene overexpression. Shownis the combined set of genes that when over expressed in NA (YPS128), WA(NCYC3290), and K11 caused a significant (FDR<0.01) fitness effect. Inblue are gene-plasmids that decreased in abundance compared to theinitial unselected pool, while in yellow are gene-plasmids thatincreased in abundance overtime compared to the initial pool.

DETAILED DESCRIPTION OF THE INVENTION

I. IN GENERAL

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention, which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); and Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell, eds., 1986).

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. Promoters that allowthe selective expression of a gene in most cell types are referred to as“inducible promoters”.

A “host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence. A host cell that has been transformed or transfected may bemore specifically referred to as a “recombinant host cell”. Preferredhost cells for use in methods of the invention include yeast cells,particularly yeast cells of the genus Saccharomyces, more preferably ofthe species Saccharomyces cerevisiae.

The nucleic acid sequence encoding MET14 is recited in SEQ ID NO: 1. Thenucleic acid sequence encoding the MDJ1 protein is recited in SEQ ID NO:2. The nucleic acid sequence encoding the ERG26 protein is recited inSEQ ID NO: 3. SEQ ID NOS:4-6 recite nucleic acids encoding therespective proteins plus native upstream promoter and downstreamterminator sequences. SEQ ID NOS:1-6 accompany this specification inAppendix A, which is incorporated by reference in its entirety.

A polypeptide “substantially identical” to a comparative polypeptidevaries from the comparative polypeptide, but has at least 80%,preferably at least 85%, more preferably at least 90%, and yet morepreferably at least 95% sequence identity at the amino acid level overthe complete amino acid sequence, and, in addition, it possesses theability to increase lignocellulosic toxin tolerance capabilities of ahost yeast cell in which is has been engineered and overexpressed.

The term “substantial sequence homology” refers to DNA or RNA sequencesthat have de minimus sequence variations from, and retain substantiallythe same biological functions as the corresponding sequences to whichcomparison is made. In the present invention, it is intended thatsequences having substantial sequence homology to the nucleic acids ofSEQ ID NO:1, 2 or 3 are identified by: (1) their encoded gene productpossessing the ability to increase lignocellulosic toxin tolerance of ahost yeast cell in which they have been engineered and overexpressed;and (2) their ability to hybridize to the sequence of SEQ ID NO: 1, 2 or3, respectively, under stringent conditions.

As used herein, “hybridizes under stringent conditions” is intended todescribe conditions for hybridization and washing under which nucleotidesequences that are significantly identical or homologous to each otherremain hybridized to each other. Such stringent conditions are known tothose skilled in the art and can be found in Current Protocols inMolecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),sections 2, 4 and 6. Additional stringent conditions can be found inMolecular Cloning: A Laboratory Manual, Sambrook et al., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. Apreferred, non-limiting example of stringent hybridization conditionsincludes hybridization in 4× sodium chlorine/sodium citrate (SSC), atabout 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. Apreferred, non-limiting example of highly stringent hybridizationconditions includes hybridization in 1×SSC, at about 65-70° C. (orhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSPE is 0.15 M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m) (° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m) (°C.)=81.5+16.6(log₁₀[Na+])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to the hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS) chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washed at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995, (oralternatively 0.2×SSC, 1% SDS).

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. As used herein, the term “polynucleotide(s)” also includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotide(s)” as that term is intended herein.Moreover, DNAs or RNAs comprising unusual bases, such as inosine, ormodified bases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

The term “isolated nucleic acid” used in the specification and claimsmeans a nucleic acid isolated from its natural environment or preparedusing synthetic methods such as those known to one of ordinary skill inthe art. Complete purification is not required in either case. Thenucleic acids of the invention can be isolated and purified fromnormally associated material in conventional ways such that in thepurified preparation the nucleic acid is the predominant species in thepreparation. At the very least, the degree of purification is such thatthe extraneous material in the preparation does not interfere with useof the nucleic acid of the invention in the manner disclosed herein. Thenucleic acid is preferably at least about 85% pure, more preferably atleast about 95% pure and most preferably at least about 99% pure.

Further, an isolated nucleic acid has a structure that is not identicalto that of any naturally occurring nucleic acid or to that of anyfragment of a naturally occurring genomic nucleic acid spanning morethan three separate genes. An isolated nucleic acid also includes,without limitation, (a) a nucleic acid having a sequence of a naturallyoccurring genomic or extrachromosomal nucleic acid molecule but which isnot flanked by the coding sequences that flank the sequence in itsnatural position; (b) a nucleic acid incorporated into a vector or intoa prokaryote or eukaryote genome such that the resulting molecule is notidentical to any naturally occurring vector or genomic DNA; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), or a restriction fragment;and (d) a recombinant nucleotide sequence that is part of a hybrid gene.Specifically excluded from this definition are nucleic acids present inmixtures of clones, e.g., as those occurring in a DNA library such as acDNA or genomic DNA library. An isolated nucleic acid can be modified orunmodified DNA or RNA, whether fully or partially single-stranded ordouble-stranded or even triple-stranded. A nucleic acid can bechemically or enzymatically modified and can include so-callednon-standard bases such as inosine, as described in a precedingdefinition.

The term “operably linked” means that the linkage (e.g., DNA segment)between the DNA segments so linked is such that the described effect ofone of the linked segments on the other is capable of occurring.“Linked” shall refer to physically adjoined segments and, more broadly,to segments which are spatially contained relative to each other suchthat the described effect is capable of occurring (e.g., DNA segmentsmay be present on two separate plasmids but contained within a cell suchthat the described effect is nonetheless achieved). Effecting operablelinkages for the various purposes stated herein is well within the skillof those of ordinary skill in the art, particularly with the teaching ofthe instant specification.

As used herein the term “gene product” shall refer to the biochemicalmaterial, either RNA or protein, resulting from expression of a gene.

The term “heterologous” is used for any combination of DNA sequencesthat is not normally found intimately associated in nature (e.g., agreen fluorescent protein (GFP) reporter gene operably linked to a SV40promoter). A “heterologous gene” shall refer to a gene not naturallypresent in a host cell (e.g., a luciferase gene present in aretinoblastoma cell line).

As used herein, the term “homolog” refers to a gene related to a secondgene by descent from a common ancestral DNA sequence. The term, homolog,may apply to the relationship between genes separated by the event ofspeciation (i.e., orthologs) or to the relationship between genesseparated by the event of genetic duplication (i.e., paralogs).“Orthologs” are genes in different species that evolved from a commonancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution. Identification of orthologs isimportant for reliable prediction of gene function in newly sequencedgenomes. “Paralogs” are genes related by duplication within a genome.Orthologs retain the same function in the course of evolution, whereasparalogs evolve new functions, even if these are related to the originalone.

The term “biofuel” refers to a wide range of fuels which are in some wayderived from biomass. The term covers solid biomass, liquid fuels andvarious biogases. For example, bioethanol is an alcohol made byfermenting the sugar components of plant materials and it is producedlargely from sugar and starch crops. Cellulosic biomass, such as treesand grasses, are also used as feedstocks for ethanol production and thepresent invention finds its primary application in this specific field.Of course, ethanol can be used as a fuel for vehicles in its pure form,but it is usually used as a gasoline additive to increase octane andimprove vehicle emissions.

“Yeasts” are eukaryotic micro-organisms classified in the kingdom Fungi.Most reproduce asexually by budding, although a few undergo sexualreproduction by meiosis. Yeasts are unicellular, although some specieswith yeast forms may become multi-cellular through the formation of astring of connected budding cells known as pseudohyphae, or falsehyphae, as seen in most molds. Yeasts do not form a single taxonomic orphylogenetic grouping. The term “yeast” is often taken as a synonym forSaccharomyces cerevisiae, but the phylogenetic diversity of yeasts isshown by their placement in separate phyla, principally the Ascomycotaand the Basidiomycota. The budding yeasts (“true yeasts”) are classifiedin the order Saccharomycetales.

The nucleotides that occur in the various nucleotide sequences appearingherein have their usual single-letter designations (A, G, T, C or U)used routinely in the art. In the present specification and claims,references to Greek letters may either be written out as alpha, beta,etc. or the corresponding Greek letter symbols (e.g., α, β, etc.) maysometimes be used.

II. THE INVENTION

This invention relates to genes that improve yeast tolerance tolignocellulosic plant hydrolysate toxins when overexpressed in yeast,preferably Saccharomyces cerevisiae. The inventors used ahigh-throughput, competitive library screen of three different yeaststrains harboring each of ˜4,500 gene overexpression plasmids, toidentify yeast genes that improve tolerance to lignocellulosichydrolysate toxins (HTs). The media is designed to mimic the sugarcontent and so-called ‘lignocellulosic’ hydrolysate toxins (HTs) foundin AFEX-treated corn stover hydrolysate (ACSH). The toxin cocktailincludes amides, weak acids, and aldehydes that have synergisticinteractions with other stresses in hydrolysate, including acetate andhigh osmolarity. The inventors identified 85 genes that improvetolerance in at least one of the three strains growing in syntheticmedium with sugar and HT content mimicking ACSH. Twenty-eight genes wereidentified in the thigh-throughput assay that improved tolerance in allthree strains. Six of these genes were validated in a single-plasmidassay: three improved strain fitness (based on end-point cell growth) intwo or three of the strains tested. These include MET14 (adenylylsulfatekinase) (SEQ ID NO:1), MDJ1 (protein folding co-chaperone) (SEQ IDNO:2), and ERG26 (C3 sterol dehydrogenase) (SEQ ID NO:3).

Accordingly, the present invention is a set of three genes that impartenhanced tolerance to toxins present in biomass hydrolysate. Theinventors have demonstrated this enhanced tolerance in a hydrolysatethat mimics the hydrolysate produced through AFEX (ammonia fiberexplosion) treatment of corn stover. The inventors identified 85 genesthat improved tolerance in at least one of the three strains growing inthe synthetic AFEX hydrolysate. Subsequent statistical screeningnarrowed the genes to twenty-eight genes that improved tolerance in allthree strains. These genes were validated in a single-plasmid assay:three improved strain fitness (based on end-point cell growth) in two orthree of the strains tested. The three S. cerevisiae strains testedincluded oak-soil isolate YPS128, which is very ligno-toxin tolerantstrain, NCYC3290 (from a West African beer fermentation) which hasmedium ligno-toxin tolerance, and sake-making strain K11 which has verylow ligno-toxin tolerance. The three genes tested that showedsignificantly improved growth in at least two of the strains are MET14(adenylylsulfate kinase), MDJ1 (protein folding co-chaperone), and ERG26(C3 sterol dehydrogenase). None of these genes has been previouslyassociated with stress tolerance in yeast. Expression of these genes inindustrial yeast strains will improve, among many advantages, theethanol yield from sugars present in AFEX hydrolysate. Presently, theinventors have used a hydrolysate mimic to select for the genes.

As can be appreciated, a major obstacle to sustainable lignocellulosicbiofuel production is microbe inhibition by the combinatorial stressesin pretreated plant hydrolysate. Chemical biomass pretreatment releasesa suite of toxins that interact with other stressors, including highosmolarity and temperature, which together can have synergistic effectson cells. Yet the combinatorial effects of such stressors as well as themechanisms cells used to survive them remain unclear. Here, theinventors explored and then exploited natural variation in stresstolerance, toxin-induced transcriptomic responses, and fitness effectsof gene overexpression to identify new genes and processes linked totolerance of hydrolysate stressors. Using six different Saccharomycescerevisiae strains that together maximized phenotypic and geneticdiversity, the inventors first explored transcriptomic differencesbetween resistant and sensitive strains to implicate common andstrain-specific responses. This comparative analysis implicated primarycellular targets of hydrolysate toxins, secondary effects of defectivedefense strategies, and mechanisms of tolerance. By dissecting theresponses to individual hydrolysate components, the inventors identifiedsynergistic interactions between osmolarity, pH, hydrolysate toxins, andnutrient composition that further inform on defense strategies.High-copy gene overexpression in three different strains revealed thebreadth of background-specific effects of gene-fitness contributions insynthetic hydrolysate. The inventors' approach identified new genes forengineering improved stress tolerance while illuminating the effects ofgenetic background on molecular mechanisms.

Based on the inventors' substantial efforts, the present inventionprovides, in a first aspect, a recombinant vector comprising: (a) thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or (b)a nucleotide sequence which hybridizes under stringent conditions to SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3 or to a fully complementarynucleotide sequence thereof; and (c) a promoter operably-linked to thenucleotide sequence of (a) or (b); wherein overexpression in yeast ofsaid nucleotide sequence provides increased tolerance to lignocellulosictoxins relative to a control yeast lacking overexpression of thenucleotide sequence.

The recombinant yeast is preferably of the genus Saccharomyces, morepreferably of the species S. cerevisiae. Such recombinant yeast willhave at least one copy of a gene which enhances toxin tolerance, and mayhave two or more, usually not exceeding about 200, depending uponwhether the construct is integrated into the genome, amplified, or ispresent on an extrachromosomal element having multiple copy numbers.Integration or non-integration may be selected, depending upon thestability required for maintenance of the extrachromosomal element, thestability of the particular extrachromosomal element prepared, thenumber of copies desired, the level of transcription available dependingupon copy number, and the like.

As used herein, the term “high copy number” when referring to arecombinant vector shall refer to a vector maintained at about 50 ormore copies per haploid genome of yeast cells.

As can be appreciated, the present invention contemplates the use ofrecombinant yeast as described herein for use in the production ofbiofuel, including certain exemplary recombinant S. cerevisiae strainsspecifically identified herein, including, as previously-described,e.g., YPS128, NCYC3290, and K11.

The present invention will, in certain embodiments, employ strongheterologous promoters, preferably inducible versions thereof. Suitablepromoters for use in the invention include, e.g., the ACT1, PGK1, TDH3,TEF1, or TEF2 promoters, or promoters of other highly expressed S.cerevisiae genes. In some embodiments, the promoter is an inducibleheterologous promoter and enhanced toxin tolerance in the recombinantyeast is conferred by induction of the inducible heterologous promoter.Inducible heterologous promoters suitable for use in the presentinvention include, e.g., the GAL4, CUP1, PHO5, or tetO7 promoter.

The present invention further encompasses a method of providing arecombinant yeast useful in biofuel production. Such a method includessteps of introducing into yeast an isolated nucleic acid having: (a) thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or (b)a nucleotide sequence which hybridizes under stringent conditions to SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or to a fully complementarynucleotide sequence thereof, wherein overexpression in the yeast of theisolated nucleic acid provides increased tolerance to a lignocellulosictoxin in the yeast relative to a control yeast lacking overexpression ofthe isolated nucleic acid.

Nucleic acid constructs useful in the invention may be prepared inconventional ways, by isolating the desired genes from an appropriatehost, by synthesizing all or a portion of the genes, or combinationsthereof. Similarly, the regulatory signals, the transcriptional andtranslational initiation and termination regions, may be isolated from anatural source, be synthesized, or combinations thereof. The variousfragments may be subjected to endonuclease digestion (restriction),ligation, sequencing, in vitro mutagenesis, primer repair, or the like.The various manipulations are well known in the literature and will beemployed to achieve specific purposes.

The various nucleic acids and/or fragments thereof may be combined,cloned, isolated and sequenced in accordance with conventional ways.After each manipulation, the DNA fragment or combination of fragmentsmay be inserted into a cloning vector, the vector transformed into acloning host, e.g., Escherichia coli, the cloning host grown up, lysed,the plasmid isolated and the fragment analyzed by restriction analysis,sequencing, combinations thereof, or the like.

Various vectors may be employed during the course of development of theconstruct and transformation of host cells. The vectors may includecloning vectors, expression vectors, and vectors providing forintegration into the host or the use of bare DNA for transformation andintegration. The cloning vector will be characterized, for the mostpart, by having a replication origin functional in the cloning host, amarker for selection of a host containing the cloning vector, may haveone or more polylinkers, or additional sequences for insertion,selection, manipulation, ease of sequencing, excision, or the like. Inaddition, shuttle vectors may be employed, where the vector may have twoor more origins of replication, which allows the vector to be replicatedin more than one host, e.g., a prokaryotic host and a eukaryotic host.

Expression vectors will usually provide for insertion of a constructwhich includes the transcriptional and translational initiation regionand termination region or the construct may lack one or both of theregulatory regions, which will be provided by the expression vector uponinsertion of the sequence encoding the protein product. Thus, theconstruct may be inserted into a gene having functional transcriptionaland translational regions, where the insertion is proximal to the5′-terminus of the existing gene and the construct comes under theregulatory control of the existing regulatory regions. Normally, itwould be desirable for the initiation codon to be 5′ of the existinginitiation codon, unless a fused product is acceptable, or theinitiation codon is out of phase with the existing initiation codon. Inother instances, expression vectors exist which have one or morerestriction sites between the initiation and termination regulatoryregions, so that the structural gene may be inserted at the restrictionsite(s) and be under the regulatory control of these regions. Ofparticular interest for the subject invention as the vector forexpression, either for extrachromosomal stable maintenance orintegration, are constructs and vectors, which in their stable form inthe host are free of prokaryotic DNA.

For extrachromosomal stable maintenance, it may be necessary to providefor selective pressure on those hosts maintaining the construct. Stablemaintenance may be achieved by providing for resistance against acytotoxic agent, e.g., an antibiotic, such as kanamycin or G418, or byimparting prototrophy to an auxotrophic host. For stable maintenance ina yeast host, the 2 micron origin of replication may be employed or acombination of a centromere, e.g., CEN3, and ars. For integration,generally homologous integration will be desirable, so that theconstruct will be flanked by at least about 50 bp, more usually at leastabout 100 bp on each side of the construct of a sequence homologous witha sequence present in the genome of the host.

The yeast host may be transformed in accordance with conventional ways.Conveniently, yeast protoplasts may be transformed in the presence of afusogen, such as a non-ionic detergent, e.g., polyethyleneglycol.

Yeast strains that may serve as yeast hosts include, for example,certain yeast strains useful in biofuel production such as, e.g.,BY4741, YB210, CEN.PK, PE-2, BG-1, CAT-1, SA-1, VR-1 or 424A(LNH-ST) andderivatives thereof. In certain yeast strains, particularly S.cerevisiae, the strains may be engineered to contain additional genessuch as, e.g., the XYL1, XYL2 and XYL3 genes of P. stipitis, which aregenerally required for most S. cerevisiae strains to ferment xylose. Ofcourse, alternative genes of roughly equal function may be used incertain embodiments; e.g., xylose isomerase (XI) may substitute forXYL1/2 in alternative embodiments, and yet other yeast strains may beengineered to include XYL1 and XYL2 genes of P. stipitis but rely onnative S. cerevisiae XYL3. Cassettes containing one or more of XYL1,XYL2 and XYL3 are available in the field. For example, XYL nucleotidesequences from P. stipitis CB56054 are available at Accession numbers:XYL1: mRNA=XM_001385144, protein=XP-001385181; XYL2: mRNA=XM-001386945,protein=XP-001386982; and XYL3: mRNA=AF127802, protein=AAF72328. Yetother embodiments may contain one or more of the CtAKR, SpNA, and SpXVT1genes useful in increased xylose fermentation.

In yet another aspect, the invention is directed to a method forproducing biofuel by fermentation of a lignocellulosic plant material inyeast, comprising: (a) culturing under biofuel-producing conditions arecombinant yeast according to the invention; and (b) isolating biofuelproduced by said recombinant yeast.

The invention further provides a method for producing biofuel byfermentation of a lignocellulosic plant material in yeast, comprising:(a) culturing under biofuel-producing conditions a recombinant yeasttransformed with a recombinant nucleic acid that overexpresses anadenylylsulfate kinase (MET14), protein folding co-chaperone (MDJ1), orC3 sterol dehydrogenase (ERG26); and (b) isolating biofuel produced bysaid recombinant yeast. The recombinant yeast is preferablySaccharomyces cerevisiae.

In certain embodiments, the recombinant nucleic acid is contained in arecombinant vector that is maintained at a high copy number in therecombinant yeast.

Methods of use according to the present invention preferably utilizelignocellulosic plant material that is an ammonia fiber explosion(AFEX)-treated lignocellulosic plant material.

Useful recombinant yeast for biofuel production methods are based on S.cerevisiae, particularly strains that have been engineered to carry oneor more of the genes described and claimed herein. Accordingly, yetanother aspect of the invention provides a recombinant Saccharomycescerevisiae strain, comprising: (a) an isolated nucleotide sequenceencoding and overexpressing an adenylylsulfate kinase (MET14), proteinfolding protein folding co-chaperone (MDJ1), or C3 sterol dehydrogenase(ERG26); (b) or a nucleotide sequence which hybridizes under stringentconditions to said isolated nucleic acid, or to a fully complementarynucleotide sequence thereof; wherein the isolated nucleotide sequence iscontained in an extrachromosomal vector maintained at a high copy numberin the strain, and said strain exhibits increased tolerance tolignocellulosic toxins relative to a control strain lacking the isolatednucleotide sequence.

In view of the various industrial uses and storage conditions thepresent recombinant yeasts will be subjected to, the invention furtherencompasses yeast inoculums which contain at least (a) a recombinantyeast engineered to contain and overexpress one or more of the isolatednucleic acids having: (a) the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; or (b) a nucleotide sequence which hybridizesunder stringent conditions to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3,or to a fully complementary nucleotide sequence thereof, whereinoverexpression in the yeast of the isolated nucleic acid providesincreased tolerance to lignocellulosic toxins in the yeast relative to acontrol yeast lacking overexpression of the isolated nucleic acid; and(b) a culture medium.

The following experimental data are provided to illustrate theinvention. It is to be understood that a person skilled in the art whois familiar with the methods may use other yeast strains, recombinantvectors, and methodology which can be equally used for the purpose ofthe present invention. These alterations are included in the scope ofthe invention.

III. EXAMPLES

In this section, the inventors describe various materials, methods andresults related to and supportive of the present invention.

Example 1. Leveraging Multi-level Natural Variation Across Saccharomycescerevisiae Isolates to Improve Lignocellulosic Hydrolysate Tolerance

This example describes a strategy to both identify strains forindustrial use and understand toxin effects by comparing and contrastingyeast strains with differential toxin tolerance. Most of the studiestrying to elucidate the mechanisms of toxin inhibition have usedlab-domesticated strains, which poorly represent the stress-tolerancepotential of the species (24-26). By studying wild isolates that displaysubstantial genetic and phenotypic differences, the inventors canidentify variation that may provide previously unrecognized modes ofprotection against toxins. Here, the inventors leveraged naturaldiversity in distinct lineages of Saccharomyces cerevisiae to explorestrain-specific responses to a synthetic mimic of ammonia fiberexpansion (AFEX)-pretreated corn stover (ACSH) (23, 27). Using asynthetic hydrolysate allowed us to dissect the transcriptional responseto the base-media composition, toxin cocktail, pH, and theircombination. Comparing strains of different resistances to thehydrolysate provided key insights into toxins' primary targets and theeffects on cellular physiology, which implicated active mechanisms toreduce toxins into less inhibitory components as well as repairingcellular damage. Due to the complex response required to survive thecombinatory effects of toxins and other stresses found in thehydrolysate, the inventors explored the role of genetic background ontoxin tolerance strategies, uncovering strain specific effects.Interestingly, the group of genes that provide fitness benefits to oneor more strains growing in synthetic hydrolysate shows low overlap withgenes whose expression responds to hydrolysate, indicating thatcombining methodologies provides a broader view of cellular defensestrategies and genetic background effects. Together, the inventors'efforts provide a glimpse into natural variation in toxin tolerancewhile implicating mechanisms and genes important for hydrolysatetolerance.

Results

Wide range of HT tolerances across Saccharomyces cerevisiae strains. Theinventors investigated the response of diverse S. cerevisiae strains tolignocellulosic hydrolysate, by phenotyping growth rates of 79 strainsgrown in base medium with and without toxins. The strains collectionincluded isolates from a variety of niches and geographical locationsand encompassed representatives of five of the defined genetic lineagesin S. cerevisiae (i.e. Malaysian, West African, North America,vineyard/European, and sake/Asian strains) (Liti et al. 2009). The groupalso included strains domesticated to ferment wine, beer, and sake,strains used to produce biofuel, and wild strains isolated from treesand spoiled fruits (Table 1). Strains were grown in a synthetichydrolysate mimic of ACSH called SynH. A phenotypic score representingresistance to hydrolysate toxins (HTs) was calculated for each strain asthe relative growth rate in complete SynH, which contains the fullcocktail of HTs, versus in the hydrolysate mimic without the toxincocktail (SynH −HTs), in both aerobic and anaerobic conditions.Resistance to HTs was highly correlated regardless of oxygenavailability (R²=0.9) (FIG. 1A); thus, the inventors focused on aerobicconditions for simplicity.

The inventors found a wide distribution of toxin-resistance phenotypes,suggesting that HT tolerance is a complex trait in yeast (FIG. 1B, Table1). Interestingly, the differences in phenotype could be partlyexplained by lineage-specific differences. The inventors grouped strainsbased on genetic lineages defined by previous population analyses of S.cerevisiae (26, 28-30). Strains of the sake/Asian lineage are the mostsensitive to HTs, while Malaysian strains display the highest resistance(FIG. 1C). Strains of the vineyard/European lineage, along with mosaicstrains that show admixture from different lineages, showed the widestdistribution of phenotypes. The lineage-specific effects are consistentwith several other studies that showed lineage-associated traits acrossstrains (24-26, 30, 31). To explore phenotypic differences in S.cerevisiae, the inventors chose six strains that would maximize thephenotypic and genetic diversity for further analysis. Five of thestrains came from clean lineages: fermentation strain NCYC361 of thevineyard/European lineage, sake-producing strain K11, West Africanstrain NCYC3290 isolated from bili wine, North American oak-tree isolateYPS128, and Malaysian strain UWO.SO5.22-7. The inventors also includedone mosaic strain Y7568, isolated from a rotten papaya, which hadparticularly high HT tolerance (FIG. 1D). Both the vineyard/European andsake strains grow slower in rich lab medium than the rest of the strains(˜90 min versus ˜70 min, doubling time); however, their growth iscomparable to well-studied lab strains ((32) and data not shown). Thephenotypes of these six strains represented the distribution seen forall strains in the collection: the sake and vineyard strains displayed˜70% decreased growth rate in the presence of HTs, the West Africanstrain displayed medium sensitivity with ˜56% decreased growth rate, andthe North American, Malaysian, and mosaic strains displayed a less-than35% decrease in growth rate when exposed to HTs.

Natural Variation in the Transcriptome Response to Lab Media ImplicatesStrain Specific States.

To explore the diverse transcriptome response among strains coming fromdifferent ecological niches, the inventors started by profilingtranscriptome differences across the six natural isolates growing inrich, non-stress laboratory medium (YPD), through RNA-sequencing inbiological duplicate. The inventors found 4,523 genes whose expressionwas significantly different (false discovery rate, FDR <1%) in one ormore strains compared to the mean of all strains, representing 72% ofall genes. Of these genes, 2,214 had at least a 2-fold expressiondifference in one or more strains compared to the mean expression levelfor that gene across all six strains.

Hierarchical clustering analysis revealed that many of thedifferentially expressed genes were specific to the HT-sensitive strainNCYC361, in which 1,200 genes were differentially expressed compared tothe mean of all strains. Expression at 858 of these genes was similarlyskewed in the HT-sensitive sake strain K11. These genes primarilydisplayed higher expression in these strains and were enriched for genesinvolved in detoxification (Bonferroni-corrected P=3.2e−8,hypergeometric test), for targets of the transcription factor Gln3 thatresponds to nitrogen limitation (P=0.001), and for thiamine genes(P=0.0004). However, unlike any other isolate, NCYC361 showed inductionof the environmental stress response (ESR) (33) even in the absence ofadded stress (P<9.83e−121). The inventors noticed that this strain hashigher expression of genes involved in iron homeostasis (P=1.2e−11) butlower expression of genes involved in the electron transport chain,amino acid biosynthesis, and lipid biosynthesis, compared to the mean ofall strains (P=4.71e−13, 2.386e−10, 8.963e−10, respectively). Thistranscriptional response can be a signature of iron starvation (34),suggesting that NCYC361 may have a defect in iron uptake/metabolism inYPD. Indeed, the inventors found that iron supplementation to YPDpartially alleviated the slow growth of this strain specifically (FIG.1).

Because specific effects in this strain obscured the broader dataset,the inventors removed NCYC361 from the analysis and performeddifferential expression among the remaining strains. The inventors found3,323 genes with significant expression differences in one or morestrains compared to the mean expression of that gene in the remainingstrains; 1,036 of these displayed at least 2-fold differences from themean. Further analysis of genes differentially expressed across strainsrevealed strain-specific transcript signatures related to thiamine,sterol, and amino acid biosynthesis among others functional responses.

Transcriptome Responses to SynH with and without HTs Implicates Commonand Strain-Specific Toxin Responses

To investigate how genetically distinct isolates experience the stressfound in lignocellulosic hydrolysate, the inventors investigatedstrains' transcriptome changes while growing in SynH compared to YPD labmedium. NCYC361 was removed from the analysis due to its aberrantresponse even in lab medium. A linear model was used to identify genesdifferentially expressed in each strain, in each media condition, and ina manner affected by a strain-by-media (“Gene by Environment”)interaction. The inventors identified 2,073 genes that weredifferentially expressed in response to SynH compared to YPD (FDR<0.01): 1,884 genes were differentially expressed regardless of thestrain, while 740 genes showed a strain-by-media interaction (FIG. 2).

Among the common responses to SynH were activation of the ESR,repression of ergosterol biosynthetic genes (P=0.023), and induction oftargets of transcription factor Sko1 that responds to osmotic stress(P=0.01). Expression of genes involved in aerobic respiration(P=7.41e−10) were increased in most strains, most highly in theHT-sensitive strain K11 and least strongly in the most resistanceMalaysian strain. The inventors also observed strong induction of genesinvolved in sulfate assimilation (P=2.46e−7) as well as a broader set ofgenes regulated by the transcription factor Gcn4, which is activated byamino acid starvation (P=1.18e−6). Higher expression of Gcn4 targetsraised the possibility that strains experienced amino acid starvation inSynH compared to rich YPD medium, especially given the stark differencesin media composition.

One key advantage of synthetic hydrolysate is that the effects ofnutrient availability and HTs can be dissected, by omitting toxins fromthe recipe. The inventors therefore profiled transcriptional changesprovoked by SynH without toxins (SynH −HTs), to distinguish the stressresponses specific to the base SynH −HT medium and responses unique tothe toxin cocktail. The inventors analyzed the response to SynH −HTscompared to YPD (omitting the wine strain NCYC361 from the analysis) andfound 970 genes differentially expressed regardless of the strain and394 genes with strain-by-media interactions (FDR <0.01).

This analysis distinguished several of responses to SynH that areprimarily due to the base medium composition separate from the toxins,and responses common to most or all strains. Genes regulated by theosmo-induced Sko1 transcription factor were induced by SynH −HTs,consistent with the high osmolarity of the base medium (FIG. 3A), whilegenes involved in ergosterol biosynthesis were repressed in response toSynH −HT medium (FIG. 3B). However, both of these responses wereexacerbated in a statistically significant manner in the presence of HTsand in several strains. This pattern was also true for genes linked tosulfate metabolism (FIG. 3C), which were induced by SynH −HTs butexpressed even higher in SynH with HTs. Thus, several responses to thebase medium were amplified by the presence of toxins, suggesting complexinteractions (see Discussion).

Other responses were specific to the presence of the toxins. Forexample, genes involved in aerobic respiration were generally expressedmore specifically in response to toxins (FIG. 3D). Surprisingly,amino-acid biosynthetic genes regulated by the Gcn4 transcription factorwere induced specifically in response to toxins and not in response tothe base SynH −HTs medium in most strains (FIG. 3E). Thus induction ofamino acid biosynthetic genes is not due to lower amino acidconcentrations in SynH but a direct response to the toxins. Addition ofamino acids, either as pools or individually, to the SynH medium did notalleviate growth inhibition (data not shown). Deleting GCN4 inHT-resistant strain YPS128 significantly reduced the growth of thatstrain but in a manner that was not specific to the presence of HTs(FIG. 9). Thus, the HT-dependent induction of Gcn4 targets may reflectan indirect response to toxins, perhaps the accumulation of unchargedtRNAs (35).

In the course of testing the effect of amino acids, the inventors foundthat strains were more sensitive to HTs at lower pH. This synergisticresponse is known for weak acids, which are significantly more toxic atlow pH because protonated acids diffuse readily into the cell (8). Totest the pH effects on other compounds, the inventors divided the HTcocktail into three groups consisting of the amides, weak acids, oraldehydes (Supplementary Table S2) and tested their inhibitory effect atpH 4.5, 5.0, or 5.5 in HT-sensitive K11 and HT-resistant YPS128. Theinventors found that low pH exacerbated the effects of all three HTclasses (FIG. 4), particularly for the HT-sensitive K11 strain (FIG.4A). In contrast, increasing pH above the normal pH of SynH improvedtolerance to weak acids and to aldehydes, but not to amides. The pHeffect was strongest when cells were exposed to the complete HTcocktail, which showed the greatest synergistic interaction with low pH,especially in HT-tolerant strain YPS128 (FIG. 4B). Thus, pH has a potentsynergistic interaction with all three classes of HTs.

Genes Responding Specifically to HTs Implicate Diverse DefenseStrategies

To explicitly identify gene expression changes to HTs, the inventorscompared the transcriptome response to SynH directly to the response toSynH −HTs across the six strains. This identified 226 genes that weredifferentially expressed in one more strains, specifically in responseto the toxin cocktail. From those genes, 149 were differentiallyexpressed independent of the strain, while 119 genes were influenced bystrain-by media interaction (FDR<0.01).

Among the induced genes were targets of the oxidant-inducedtranscription factors Yap1 (P=1.5e−14) and Skn7 (P=7.4e−5), consistentwith the notion of redox stress induced by the HTs cocktail. Many of theother induced genes include those that are induced in response to abroad array of stresses (33). These included genes encoding heat shockproteins and genes responding to high osmolarity, cell wall integrity,DNA damage response, and organic solvent stress. Consistently, inducedgenes were enriched for known targets of the general stresstranscription factor Msn2 that responds to a wide array of stresses(P=0.0014). The induced gene set also included several genes that areimplicated in the reduction of HTs into less toxic compounds, and theseincluded aldehyde reductases and dehydrogenases, aryl alcoholdehydrogenases known to be involved in oxidative stress response (36),an alpha-keto amide reductase most likely responding to the toxic amidesin the HTs cocktail, and plasma membrane transporters involved in toxintransport among others (Table 3).

Interestingly, genes related to thiamine metabolism were enriched(P=0.000123) in the set of HT-responsive genes having lower expressionin several strains. These included four genes involved in thiaminebiosynthesis (Thi2, Thi6, Thi20, Thi21) and two genes involved inthiamine uptake (Thi7, Thi73). Thiamine is important for sugarfermentation (37) and has also been shown to play a role in the defenseagainst oxidative and osmotic stress in S. cerevisiae (38), thus thisresult was unexpected. Recently, it was discovered that the expressionof thiamine genes can respond to NAD+ levels (39), since NAD+ is aprecursor for de novo thiamine production (40). The inventors reasonedthat the response of thiamine genes could reflect fluctuations inNAD+/NADH levels during HTs detoxification. The inventors quantifiedNAD⁺/NADH in the absence and presence of HTs. Strikingly, the inventorsfound that total NAD+ plus NADH (NAD+/H) levels (FIG. 5A) as well as theNAD+/NADH ratio (FIG. 5B) increased in the presence of HTs. This wastrue for both the resistant strain YPS128 and the sensitive K11 strain,although the effect was greater in K11. Interestingly, genes involved inde novo biosynthesis of NAD+ were also induced by the presence of HTs.Together, these results suggest that cells increase levels of NAD+/H inthe presence of toxins, perhaps reflecting a defect in NADH regenerationduring the course of detoxification.

Identifying Genes Whose Expression Correlates with HT Resistance AcrossStrains

The inventors were especially interested in exploiting the physiologicaldifferences between resistant and sensitive strains to find novel genesand mechanisms that could increase SynH tolerance. The inventorstherefore identified genes whose expression level was correlated withtoxin tolerance (see Methods). This identified 253 genes whoseexpression was negatively correlated with HT resistance (FIG. 6A) and 32genes whose transcript abundance was positively correlated with HTresistance (FIG. 6B). The genes whose transcript abundance wasnegatively correlated with HT resistance—meaning that they wereexpressed proportionately higher as HT tolerance decreased—suggestedcellular targets of the toxins. Although enrichment did not passstringent bonferroni correction, 21% of these genes encode proteinslocalized in the mitochondria (uncorrected p=0.0006). This groupincluded other genes involved in cell wall organization, fatty acidmetabolic process, DNA repair, protein folding, and genes involved inNAD biosynthesis. The stronger expression response in HT-sensitivestrains suggests that cells experiencing stronger HT stress may strugglemore to maintain critical processes. Consistent with this notion,sensitive cells generally showed higher expression of genes induced inthe Environmental Stress Response than tolerant cells (FIG. 10). Incontrast, genes whose expression was positively correlated with HTresistance were enriched for translation (uncorrected p=1.4e−7),suggesting that resistant cells may be growing better under theseconditions.

Other genes whose expression was positively correlated with HTresistance were involved in fatty acid elongation (uncorrectedp=6.8e−5), amino acid transmembrane transport (uncorrected p=0.005), anddegradation of arginine (uncorrected p=2.2e−5).

Fitness Effects of Gene Overexpression are Influenced by GeneticBackground

The inventors were particularly interested in identifying and testinggenes whose overexpression improved HT tolerance. To do this, theinventors measured changes to cellular fitness due to high-copyexpression of each of 4,282 genes, using ‘bar-seq’ analysis of ahigh-copy gene library expressed in three different strains (YPS128,NCYC3290, and K11) growing in media with HTs (see Methods).

The effects of gene overexpression were significantly influenced bygenetic background (FIG. 11). Of all genes that increased fitness inSynH in any strain, only 32% (28 genes) were common in all threestrains. These were weakly enriched for genes annotated in mRNAlocalization (uncorrected P=0.001) and cellular carbohydrate metabolicprocess (uncorrected P=0.002) (FIG. 7A). Somewhat surprisingly, thetolerant strain YPS128 showed fitness increases in response to thegreatest number of overexpressed genes (which together had weakenrichment for genes encoding membrane proteins, uncorrected P=0.006),while the sensitive sake strain was influenced by only a singlestrain-specific gene (DBP2, whose functions involved mRNA decay and rRNAprocessing (41, 42), although the inventors cannot exclude that thesetrends are not influenced by differences in statistical power (seeMethods). Interestingly, the sensitive sake strain showed a fitnessdefect in response to overexpression of a large number of genes (FIG.7B), with weak enrichment for GTPase activator activity (un-correctedP=0.0003), SNARE binding (un-corrected P=0.0005), and ubiquitin-proteinligase activity (un-corrected P=0.0005). The extensive differences infitness contributions depending on strain background highlights thatstrategies for engineering tolerance to a complex stress such as theones found in SynH may require strain-specific strategies.Interestingly, there was no statistically significant overlap in thehigh-copy genes that contributed fitness benefits to one or more strainsand genes that showed significant expression differences across strains.2 out of 28 genes identified in the overexpression study had asignificant expression change specifically in response to HTs.

The inventors confirmed the library results by measuring fitness ofcells expressing individual plasmids, compared to the empty-vectorcontrol (FIG. 7B). The inventors chose three genes identified in allstrains (MET14, THI20, ERG26), two genes identified in two of thestrains (MDJ1, identified in YPS128 and NCYC3290, and TPK2, identifiedin YPS128 and K11), and one gene (NUP53) specific to the tolerant strainYPS128. MET14 and THI20 were identified as having differentialexpression in HT presence (FIG. 7). The inventors also included as acontrol one gene (PBI1) whose expression was not predicted to changefitness. Among the six tested genes, three of them (MET14, ERG26, andMDJ1) significantly improved growth in at least two strains. The moststriking was MDJ1, involved in protein folding/refolding in themitochondrial matrix (43), which improved growth 118% in NCYC3290 and28% in the already-tolerant YPS128. Most of the genes did not provide astrong benefit over the empty vector in K11; however, the strongnegative impact of thousands of genes in the library suggests that thisstrain has a competing fitness deficit due to protein overexpression.The inventors note that this assay, comparing the effects of individualplasmids to the empty-vector control, is different from the competitivelibrary experiment, in which each gene's fitness contribution iseffectively normalized to the average of all plasmids (see Methods).

Discussion

In real industrial fermentations, multiple distinct stresses can havecompounded effects that produce unique challenges for cells. How thesedifferent stressors interact with one another can be difficult todiscern, especially in real hydrolysates that can vary extensively frombatch-to-batch and according to the biomass type and source (44, 45).Furthermore, the response can be quite different depending on thegenetic background of the strain. These distinctions present challengesfrom an industrial standpoint, especially in terms of identifyinggeneral mechanisms to improve tolerance to industrial stresses.

Our strategy to leverage natural variation, both to understand stressorsin lignocellulosic hydrolysate and to identify high-impact genes fordirected engineering, presents a useful strategy to tackle thesehurdles. Responses common to all strains implicated the imposingstresses in SynH, including osmotic stress from the high sugarconcentrations, oxidative stress produced by several HTs (46, 47), andredox imbalance, perhaps due to HT detoxification (48, 49). In contrast,responses that were graded with HT sensitivity implicate downstreamcellular targets at greatest risk. For example, sensitive strainsdisplayed stronger expression changes at genes involved in cell wallorganization, fatty acid metabolism, DNA repair, protein folding,suggesting that the cell wall, membranes, the genome and proteome areprimary targets of the reactants in the HT cocktail (12, 17, 49, 50).The more sensitive strains also had stronger induction of genes involvedin energy generation, suggesting a tax on the energy balance (48, 51,52). Our results also implicate a variety of defense strategies,including toxin reduction, redox defense, and drug efflux anddetoxification. Several of these strategies required NADH (53-58), whichlikely contributes to the observed increase in NAD+/H and the NAD+/NADHratio across strains (a response also seen in Escherichia coli growingin SynH (51)).

Comparing strain responses under different situations also revealed newinsights into synergistic stress interactions. HT sensitivity wasexacerbated at low pH, expanding the know synergy between pH and weakacids (8) to interactions with other HTs and in particular the full HTcocktail. Several expression responses to SynH −HT were exacerbated bythe addition of HTs. In some cases, dual stressors may exacerbate asingle cellular challenge. For example, the amplified induction ofsulfur biosynthesis genes when HTs are added to the base medium may be aresponse to NADPH depletion, since both HT detoxification and sulfurassimilation consume NADPH (13, 14, 59). Notably, sulfate genes are alsoinduced in bacteria growing in the presence of furfural (59) and in SynH(51). In other cases, the synergy may emerge because the defensestrategy against one stress renders cells more sensitive to a secondstressor. For example, cells growing in high-osmolarity SynH −HTsinduced expression of osmo-induced Sko1 targets (60) and decreasedexpression of ergosterol biosynthesis genes. Decreased ergosterol is aphysiologically adaptation to osmotic stress that may help to decreasemembrane fluidity (61-63). However, reduced ergosterol is associatedwith lower resistance to vanillin (64); thus, altered ergosterol contentcould produce antagonistic effects on tolerance to osmolarity versusHTs. It is particularly interesting that genes related to two of theseinteractions—adenylylsulfate kinase MET14 required for sulfurassimilation and ERG26 involved in ergosterol synthesis—improve HTtolerance in the context of high sugar concentrations, in all threestrains tested.

It is well known in the industry that engineering strategies are strainspecific (65, 66); yet most investigations fail to consider this whenidentifying new engineering targets and instead focus on a single, oftenlaboratory, strain. Our approach to examine multiple strains thattogether maximize genetic and phenotypic diversity not only implicatedgenes with background-independent benefits, but also uncovered thebreadth of responses in the species. Over half the mRNAs in thetranscriptome varied in abundance across strains, in one or moreconditions. In several cases, the inventors were able to predict andvalidate cellular phenotypes based on transcriptomic differencesdemonstrating how far knowledge of yeast gene functions has progressedin terms of predictive power. But in other ways, our results highlightthe limitations in understanding the interaction between genotype andphenotype. This is particularly true in the case of high-copy geneexpression, whose differential effects suggest that background effectswill be the norm rather than the exception. Our work sets the stage formore detailed mapping of phenotypic variation across strain backgrounds.

Materials and Methods

Strains and Growth Condition

Strains and phenotypes are listed in Table 1. The SynH media mimics ACSHwith 90 g glucan/L loading and was prepared as in Serate et al. (2015)except that all concentrations were increased 1.5-fold to emulate ahigher glucan loading (Table 2). Gene knockouts were generated byhomologous recombination of the KAN-MX cassette into the locus ofinterest in a haploid version of YPS128 and verified by diagnostic PCR.Unless otherwise indicated, cultures were grown with vigorous shaking at30° C. Where indicated, media was supplemented with iron (II) sulfateheptahydrate (Sigma-Aldrich, St. Louis, Mo.). Overexpression experimentswere performed using the molecular barcoded yeast (MoBY 2.0) ORF library(67), growing cells in Synthetic Complete medium (SC) with high sugarconcentrations and no ammonium to support G418 selection (68) (1.7 g/LYNB w/o ammonia sulfate and amino acids, 1 g/L monosodium glutamic acid,2 g/L amino acid drop-out lacking leucine, 48 μg/L leucine, 90 g/Ldextrose, 45 g/L xylose) along with the toxin cocktail (Table 4).

TABLE 1 HT relative Strain source Location of growth Strain categoryisolation rate NCY3455 Clinical Newcastle, UK 5 K9 Sake Japan 10 UC5Sake Kurashi, Japan 15 Y2189 Natural Isolate San Jacinto, 24 California,USA K10 Sake Japan 26 NCYC361 Other Fermentation NA 29 YJM269 OtherFermentation NA 30 YB4082 Natural Isolate Philippines 31 Y1 NaturalIsolate NA 32 YJM320 Clinical USA 34 K11 Sake Japan 34 K1 Sake Japan 35SK1 Lab USA 37 YJM428 Clinical USA 38 322134S Clinical Newcastle, UK 38SB Natural Isolate Indonesia 40 YS2 Bakery Australia 42 NCY3290 OtherFermentation Indonesia 44 L-1528 Wine fermentation Maule Region, 47Chile DBVPG6765 Natural Isolate Indonesia 48 DCM6 Natural IsolateWisconsin, USA 49 Y389 Natural Isolate NA 49 CLIB324 Bakery Vietnam 49273614N Clinical Newcastle, UK 50 YPS1009 Oak Mettlers Woods, 50 NJ, USACLIB382 Other Fermentation Ireland 51 WE372 Wine fermentation Cape Town,South 52 Africa YJM308 Clinical USA 53 M22 Vineyard Italy 54 T73 Winefermentation Alicante, Spain 54 378604X Clinical Newcastle, UK 54DBVPG6044 Other Fermentation West Africa 54 DBVPG1853 Unknown Ethiopia57 YJM326 Clinical USA 58 CBS7960 Industrial Sau Paulo, Brazil 58fermentation (sugar cane) YJM978 Clinical Bergamo, Italy 58 YPS606Natural Isolate Woodland, PA 59 Y6 Natural Isolate French Guiana 59DBVPG1373 Natural Isolate The Netherlands 59 YJM981 Clinical Bergamo,Italy 60 DBVPG1788 Natural Isolate Turku, Finland 60 YPS163 OakPennsylvania, USA 60 YB210 Natural Isolate Costa Rica 60 YJM454 ClinicalUSA 60 YPS1000 Oak Mettlers Woods, 60 NJ, USA YIIc17_E5 Winefermentation Sauternes, France 61 YJM1129 Other Fermentation NA 61DBVPG1106 Natural Isolate Australia 61 Y2 Other Fermentation Trinidad 61YJM975 Clinical Bergamo, Italy 62 UWOPS83-787.3 Natural Isolate Bahamas62 BC187 Other Fermentation Napa Valley, USA 63 I14 Vineyard Petina,Italy 63 CLIB215 Bakery New Zealand 63 YS9 Bakery Singapore 63 DCM16Natural Isolate Wisconsin, USA 63 Y3 Other Fermentation Africa 64 YS4Bakery The Netherlands 64 YJM339 Clinical USA 64 IL-01 NA Cahokia, IL 64YJM421 Clinical USA 64 NCYC110 Other Fermentation West Africa 65 Y55 LabNA 65 L-1374 Wine fermentation Maule Region, 65 Chile YPS128 NaturalIsolate Pennsylvania, USA 66 YJM440 Clinical USA 66 NC-02 OtherFermentation Smoky Mountains, 67 NC UWOPS87-2421 Natural isolate Hawaii68 Y7568 Natural Isolate Philippines 68 FL100 Lab NA 69 PW5 OtherFermentation Aba, Nigeria 69 PE-2 Other Fermentation Brazil 69EthanolRed Other Fermentation NA 70 YJM653 Clinical NA 71 Y2209 NaturalIsolate San Jacinto, 72 California, USA YJM451 Clinical Europe 73UWOPS05-227.2 Natural Isolate Trigona, Malaysia 74 UWOPS05-217.3 NaturalIsolate Malaysia 75 UWOPS03-461.4 Natural Isolate Malaysia 78

TABLE 2 HT groups AMIDES ACIDS ALDEHYDES Feruloyl amide p-Coumaric acidVanillin Coumaroyl amide Ferulic acid Syringaldehyde Benzoic acid4-Hydroxybenzeldehyde Syringic acid 4-Hydroxyacetophenone Cinnamic acidHydroxymethyl furfural Vanillic acid Caffeic acid

TABLE 3 PROCESS GENES Oxidation-reductase activity Quinone reductaseZTA1, YLR460c Aldehyde reductase ARI1 Alcohol dehydrogenase ADH7, ADH5Alpha keto amide reductase YDL124W Aryl alcohol dehydrogenase AAD4,AAD6, AAD16 Nitric oxide oxidoreductase YHB1 Oxidation of thiols FMO1Nitroreductase FRM2 Fatty-acyl coenzyme A oxidase POX1 NADPHoxidoreductase OYE3 NADPH regeneration YMR315W, PYC1, ZWF1 de-novo NADbiosynthesis from tryptophan BNA1, BNA5 Lyase activity DecarboxylaseFDC1 Plasma Membrane Transporter ABC Transporter SNQ2, PDR12 Multidrugtransporter FLR1

TABLE 4 SynH base media Component mM Final Concentration KH₂PO₄ 8.76K₂HPO₄ 16.725 (NH₄)₂SO₄ 45 KCl 55.2 NaCl 1.95 CaCl2•2H2O 8.25 MgCl2•6H2O18.75 L-Alanine 1.758 L-Arginine•HCl 0.216 L-Asparagine 0.342DL-Aspartic acid•K 0.891 L-Cysteine•HCl 0.075 L-glutamine 0.3885L-Glutamic acid•K 0.9105 Glycine 0.567 L-Histidine 0.0561 L-Isoleucine0.393 L-Leucine 0.5565 L-Lysine•HCl 0.2625 L-Methionine 0.15L-Phenylalanine 0.423 L-Proline 0.984 L-Serine 0.5535 L-Threonine 0.465L-Tryptophan 0.075 L-Valine 0.636 L-Tyrosine 0.303 Adenine 0.075Cytosine 0.075 Uracil 0.075 Guanine 0.075 Thiamine HCl 0.0006 CalciumPantothenate 0.0045 ZnCl₂ 30 MnCl₂•4H₂O 136.5 CuCl₂•2H₂O 2.85 CoCl₂•6H₂O0.045 H₃BO₄ 34.65 (NH₄)₆Mo₇O₂₄•4H₂O 0.465 FeCl3•6H2O 30 Sodium formate4.2 Sodium nitrate 1.65 Sodium succinate 0.75 Glycerol 6.15 Betaine•H2O1.05 Choline Chloride 0.45 DL-Carnitine 0.45 Acetamide 120 Sodiumacetate 48 L-lactatic acid (90%) 6 D-Mannose 1.8 L-Arabinose 30D-Fructose 36 D-Galactose 4.35 D-Glucose 90 g/l (500 mM) D(+)Xylose 45g/l (300 mM) Pyridoxine•HCl 3.21 μM Nicotinic Acid 40.17 μM Biotin 0.15μM Inositol 0.084 mM Polysorbate 80 (Tween 80) 1.5 ml/l Ergosterol 15mg/l

TABLE 5 HT cocktail Toxin mM Feruloyl amide 8.25 Coumaroyl amide 8.255-Hydroxymethyl-2furadehyde 1.65 p-Coumaric acid 3.15 Ferulic acid 1.065Benzoic acid 0.73 Syringic acid 0.11 Cinnamic acid 0.14 Vanillic acid0.13 Caffeic acid 0.02 Vanillin 0.20 Syringaldehyde 0.244-Hydroxybenzeldehyde 0.30 4-Hydroxyacetophenone 0.04Phenotyping

10 μl of thawed frozen stock of cells was used to inoculate a 96-wellplate (NUNC, Thermo Scientific, Rockford, Ill.) containing 190 μl of YPDmedia. Plates were sealed with breathable tape (AeraSeal, Sigma-Aldrich,St. Louis, Mo.), covered with a lid and incubated at 30° C. whileshaking for 24 h, after which 10 μl of saturated cultures were used toinoculate 190 μl of YPD and grown to log phase for 6 h. Growthphenotyping was performed after inoculating 10 μl of the log phaseculture into 190 μl of SynH or SynH −HTs, and growing without shaking inTecan M200 Pro microplate reader (Tecan Systems, Inc., San Jose, Calif.)maintaining an interior chamber temperature of 30° C. Anaerobicphenotyping was performed similarly using a Tecan F500 inside ananaerobic chamber. The average of six optical density at 600 nm (OD₆₀₀)measurements distributed from across the well was taken every 30 minutesfor 48 hours. Growth rates were calculated using the program GrowthRates(69). An HT resistance score was taken as the average of twobiological-replicate growth rate measurements from SynH versus theaverage growth rates in SynH −HTs, in both aerobic and anaerobicconditions.

RNA-Seq Library Construction and Sequencing

Strains were grown in biological duplicate on different days to mid-logphase for seven generations in YPD and then shifted to YPD, SynH −HTs,or SynH medium for at least three generations to log phase (OD600 ˜0.5)and collected by centrifugation. RNA was extracted by hot phenol lysis(70). Total RNA was DNAse-treated at 37° C. for 30 min with TURBO DNase(Life Technologies, Carlesbad, Calif.), followed by RNA precipitation at−20° C. in 2.5M LiCl for 30 min. rRNA depletion and library generationwas via the TruSeq® Stranded Total RNA Sample Preparation Guide (Rev.C)using the Illumina TruSeq® Stranded Total RNA (Human/Mouse/Rat) kit(Illumina Inc., San Diego, Calif., USA) with minor modifications, usingAgencourt RNAClean XP beads (Beckman Coulter, Indianapolis Ind., USA),SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, Calif., USA)as described in the Illumina kit. Adapter ligated DNA was amplified in aLinker Mediated PCR reaction (LM-PCR) for 12 cycles using Phusion™ DNAPolymerase and Illumina's PE genomic DNA primer set and then purified byparamagnetic beads. Libraries were standardized to 2 μM. Clustergeneration was performed using standard Cluster Kits (v3) and theIllumina Cluster Station. Single-end 100 bp reads were generated usingstandard SBS chemistry (v3) on an Illumina HiSeq2500 sequencer. Raw datawas deposited in NIH SRA database under project number GSE77505.

RNA-Seq Read Processing and Analyses

Reads were processed with Trimmomatic (71) and mapped to referencegenome S288C (NC-001133, version 64 (72)) using Bowtie2 (73) withdefault settings. HTseq version 5.5 (74) was used to calculate readcounts for each gene. Differential expression analysis was performedusing the program edgeR v.3.8.6 (75) using a general linearized modelwith strain background and media type as factors and pairing replicatesamples. Benjamini and Hochberg correction (76) was used to estimateFDR. Sequences were normalized using the reads per kilobase per millionmapped reads (RPKM) method. Hierarchical clustering analysis wasperformed using the program Cluster 3.0 (77) and visualized with theprogram Java Treeview (78). Where noted, expression of each gene wasnormalized to the mean expression level for that gene across allstrains. Functional enrichment analysis was performed using FunSpec (79,80). All P values cited are Bonferroni-corrected, unless otherwisenoted.

NAD+/NADH Measurement

Total NAD and NAD⁺/NADH were measured in biological triplicate usingQuantification Colorimetric Kit (BioVision, Milpitas, Calif.) followingthe recommended protocol. Briefly, strains were grown in SynH and SynH−HTs for at least three doublings, and collected while in log phase(OD600 ˜0.5). NAD and NADH levels were calculated as outlined by thekit, and NAD+ was inferred from the other two measurements.

Correlations Between Expression and Toxin Tolerance

The inventors first identified 2,777 genes with significant expressiondifferences compared to the mean expression for that gene (FDR<0.01).The inventors then averaged the replicate RPKM expression values foreach strain and used Python statistical functions (SciPy.org) tocalculate the Pearson correlation between each gene's expression patternand the HT resistance scores across strains. Genes whose expressioncorrelated with resistance were chosen based on p<0.05.

High Throughput Gene Overexpression Fitness Effects

Competition experiments were performed similar to that previouslydescribed (81, 82). Briefly, a molecular barcoded yeast ORF library(MoBY-ORF 2.0) (67, 83) was introduced into three different strains, bytransforming cells with a pool library of the MoBY 2.0 collectioncontaining 4,282 barcoded high-copy plasmids, each expressing adifferent yeast gene. Transformation efficiency was determined byplatting serial dilutions onto YPD agar+G418-containing plates.Transformations with more than 30,000 colonies were pooled together togenerate glycerol stocks used for further experiments. For competitionexperiments, cells were grown in SC containing monosodium glutamate as anitrogen source and high sugar mimicking SynH (9% glucose, 4.5% xylose)plus HT cocktail and 200 mg/L of G418 (see Media) for 5, 10, and 15generations, while maintaining cells in log phase. This medium was usedinstead of SynH since G418 selection required for plasmid maintenancedoes not function in the presence of ammonium. DNA was extracted usingQIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) after cell pelletpretreatment with 1 μl of RZymolyase (Zymo Research, Irvine, Calif.) and100 μl of glass beads, with vortexing for 5 minutes. Plasmid barcodeswere amplified with multiplex primers containing Illumina adapters.Barcodes of two replicates were sequenced using an Illumina HiSeq2500Rapid Run platform. Differential abundance and significance of plasmidswere determined using edgeR (75), using a linear model for each strain,identifying genes that provided a significant different fitnesscontribution to media +HTs compared to the starting pool beforeselection over time (5, 10, and 15 doublings).

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As can be appreciated, the results described in the above examplessupport the utility of the nucleic acids, yeast strains and methodsdescribed and claimed herein for enhancing biofuel production in yeast.Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific materials, methods, formulations, reaction/assayconditions, etc., herein illustrated and described, but embraces suchmodified forms thereof as come within the scope of the following claims.

What is claimed is:
 1. A recombinant vector comprising: (a) thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and (b)a promoter operably-linked to the nucleotide sequence of (a); whereinoverexpression in yeast of said nucleotide sequence provides increasedtolerance to lignocellulosic toxins relative to a control yeast lackingoverexpression of the nucleotide sequence.
 2. The recombinant vector ofclaim 1, wherein said vector includes heterologous nucleotide sequencesthat stably maintain the vector at a high copy number when transformedinto yeast.
 3. The recombinant vector of claim 1, wherein the promoteris a heterologous promoter.
 4. A recombinant yeast comprising therecombinant vector of claim
 1. 5. The recombinant yeast of claim 4,wherein the recombinant yeast is of the genus Saccharomyces.
 6. Therecombinant yeast of claim 5, wherein the recombinant yeast is of thespecies Saccharomyces cerevisiae.
 7. The recombinant yeast of claim 4,wherein the recombinant vector is an extrachromosomal vector stablymaintained in the recombinant yeast.
 8. The recombinant yeast of claim4, wherein the recombinant vector is stably maintained at a high copynumber in the recombinant yeast.
 9. The recombinant yeast of claim 4,wherein the recombinant vector is integrated into a chromosome of therecombinant yeast.
 10. A recombinant Saccharomyces cerevisiae strain,comprising: an isolated nucleotide sequence encoding and overexpressingan adenylylsulfate kinase (MET14), protein folding protein foldingco-chaperone (MDJ1), or C3 sterol dehydrogenase (ERG26); wherein theisolated nucleotide sequence is contained in an extrachromosomal vectormaintained at a high copy number in the strain, and said strain exhibitsincreased tolerance to lignocellulosic toxins relative to a controlstrain lacking the isolated nucleotide sequence.